PNAS: New Study Shows that Remdesivir Prevents Coronavirus MERS-CoV Infection in Monkeys

In a new study, researchers from the National Institutes of Health reported that the experimental antiviral drug remdesivir (also known as GS-5734) successfully prevented rhesus monkeys infected with the Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV) from becoming ill from this virus infection. Giving remdesivir before infection can prevent them from getting sick, while giving this drug after they are infected can improve their condition. The results were published online February 13, 2020 in the journal PNAS, entitled “Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection”.

 

MERS-CoV is closely related to the 2019 novel coronavirus (SARS-CoV-2, also known as COVID-19, previously known as 2019-nCoV). Since the first case of 2019-nCoV infection was detected in Wuhan, China in December 2019, this virus has triggered a large-scale epidemic.

 

Remdesivir has previously been shown to protect animals from multiple viruses in laboratory experiments. Experiments have confirmed that the drug is effective in treating monkeys infected with Ebola virus and Nipah virus. It has also been studied in humans for the treatment of Ebola virus disease.

 

This new study involved three groups of monkeys: monkeys that were administered remdesivir 24 hours before infection with MERS-CoV; monkeys that were administered remdesivir 12 hours after infection (close to the peak time of MERS-CoV replication in these monkeys); and monkeys that were not administered remdesivir (as a control group).

 

The researchers observed the monkeys for six days. All control monkeys showed signs of respiratory disease. The monkeys that were given remdesivir before infection were in good health: they had no signs of respiratory disease, had markedly decreased levels of viral replication in the lungs, and had no lung damage compared with control monkeys. Monkeys that were given remdesivir after infection performed much better than control monkeys: the severity of the disease was lower than control monkeys, their lung virus levels were lower than control monkeys, and their lung damage was also milder.

 

These researchers point out that these promising findings support the use of remdesivir for clinical trials against MERS-CoV and 2019-nCoV. In China, several clinical trials are ongoing to treat patients with 2019-nCoV using remdesivir.

 

Gilead Sciences Inc. Developed the remdesivir and participated in this study.

 

In 2012, MERS-CoV emerged in Saudi Arabia. By December 2019, the World Health Organization had identified 2499 MERS-CoV cases and 861 deaths. Given that about one third of MERS-CoV cases are transmitted by infected individuals treated at health facilities, these researchers propose that remdesivir may be effective in protecting other patients, patient contacts, and health care workers from disease. They also noted that if this medication is taken shortly after the onset of symptoms, then it may help patients with a confirmed diagnosis of MERS-CoV or 2019-nCoV.

Creative Proteomics Scientists Share Precautions for Chromatin Immunoprecipitation Experiments

Eukaryotic genomic DNA exists as chromatin. Therefore, studying the interaction between protein and DNA in chromatin environment is the basic way to elucidate the gene expression mechanism of eukaryotes. Chromatin immunoprecipitation (CHIP) is currently the only method to study the interaction between DNA and proteins in vivo.

Its basic principle is to fix the protein-DNA complex in a living cell state and randomly cut it into small chromatin fragments within a certain length range, and then precipitate this complex by immunological methods to specifically enrich the target protein The bound DNA fragments are used to purify and detect the target fragments to obtain information about protein-DNA interactions.

CHIP can not only detect the dynamic effects of trans factors and DNA in vivo, but also can be used to study the relationship between various covalent modifications of histones and gene expression. In addition, the combination of CHIP and other methods has expanded its application scope. The CHIP-on-chip method established by combining CHIP and gene chips has been widely used for high-throughput screening of specific trans factor target genes; CHIP with in vivo footprinting methods is used to find the binding site of trans factor in vivo; RNA-CHIP is used to study the role of RNA in the regulation of gene expression. It can be seen that with the further improvement of CHIP, it will play an increasingly important role in the study of gene expression regulation.

Chromatin Immunoprecipitation (ChIP) is a method developed based on in vivo analysis, also known as binding site analysis. It has become the main method of epigenetic information research in the past decade. This technology helps researchers determine what histone modifications will occur at a particular location in the genome of the nucleus.

ChIP can not only detect the dynamic effects of trans factors and DNA in vivo, but also can be used to study the relationship between various covalent modifications of histones and gene expression. In recent years, this technology has been continuously developed and improved. Combining microarray technology to examine chromosomal activity in chromosomal gene expression regulatory regions is a very effective tool for in-depth analysis of major metabolic pathways for diseases such as cancer, cardiovascular disease, and central nervous system disorders.

Its principle is that while maintaining the combination of histone and DNA, through the use of a biological antibody corresponding to a specific histone tag, chromatin is cut into very small pieces and precipitated. IP is a method developed by utilizing the specific binding of an antigen protein and an antibody, and the phenomenon that “prorein A” of a bacterial protein specifically binds to an FC fragment of an immunoglobulin. At present, the multi-purpose refined prorein A is pre-bound and solidified on the beads of argarose, and after reacting with the solution and antibody containing the antigen, the prorein A on the beads can adsorb the antigen to achieve the purpose of purification.

The most important point of the experiment is the nature of the antibody. Different antibodies and antigen-binding capabilities are different, and dye-free binding may not be used in IP reactions. It is recommended to check the antibody manual carefully. In particular, the specificity of polyclonal antibodies is a problem.

Secondly, pay attention to the nature of the buffer in which the antigen is dissolved. Most antigens are cellular proteins, especially backbone proteins, which must be lysed by the buffer. For this reason, a buffer containing a strong surfactant must be used, although it may affect the binding of a part of the antigen-antibody.

On the other hand, if the cells are lysed with a weak surfactant, the cell proteins cannot be fully lysed. Even the dissolution also results in binding to other proteins, the epitope is blocked, affecting the binding to antibodies, and even if IP is successful, it is a tragic result of co-precipitation of many proteins and antibodies.

Third, in order to prevent protein degradation and modification, the antigen-dissolving buffer must be added to each inhibitor of the protein, and the experiment should be performed at low temperature. Before each experiment, first consider the antibody / buffer ratio. Too little antibody will not detect the antigen, too much will not settle on the beads, and the supernatant will remain. Too little buffer will not dissolve the antigen, too much will dilute the antigen.

Creative Proteomics can provide custom experimental design and refine Co-IP parameters based on customers’ needs. The company promises reliable Co-IP analysis of protein-protein interactions with its experienced scientists and technicians. Established in 2004, Creative Proteomics has gradually developed into an integrated company that provides proteomics, metabolomics, glycomics, and bioinformatics analysis services to researchers in the pharmaceutical, biotechnology, agriculture and nutrition industries, as well as academic and government organizations.

 

TOR: The secret to maintaining longevity and healthy aging

From yeast to monkeys, calorie-restricted diets have been shown conducive to life extension and healthy life of all living organisms as long as there is no malnutrition. Although there is no long-term research to prove the benefits of limiting calories to human life, short-term studies have shown that it does improve health.

 

It might work like this. Our body monitors and senses the amount of nutrients through specific molecules in the cell. Depending on the amount of food we eat, these molecules regulate our metabolism to intervene how we use the available nutrients. One of these molecules is an enzyme called TOR. When there are many foods, the TOR enzyme will indicate cells to grow in the body. If there is less food, TOR will indicate cells to be vigilant – what scientists call “mild stress response.”

 

Many experiments have shown that when animals eat a lot of food, especially for a long time, TOR will feel this, and their life will be shorter. But does all food have this effect on TOR?

 

The TOR enzyme is particularly active when cells sense large amounts of amino acids (components of proteins) or proteins. Protein-restricted diets do not cause malnutrition and can have the same effects on the metabolism and longevity of laboratory animals as calorie-restricted diets.

 

Age-related diseases

 

It is well known that age-related diseases are caused by genetic mutations, but is there a link between TOR, nutrition and senile diseases? We know that nutrition is associated with cancer and heart disease, and overactive TOR is associated with these diseases, but recent studies have shown that TOR is also directly associated with neurodegenerative diseases. For example, the TOR activity in the brain of Alzheimer’s patients is much higher than in a healthy brain. In addition, mimicking these diseases in mice and other experimental animals suggests that removal of excess TOR can prevent brain cell death.

 

So, what we eat, how our body perceives, and the risk of neurodegenerative diseases may be linked. Scientists are exploring various possibilities to prevent neurodegeneration. If more protein means more active TOR, we can either safely adjust our diet or develop a drug that deceives our body and makes it think it consumes less protein.

 

Many laboratory studies have shown that caffeine and a drug called rapamycin actually can do that. Although cells are rich in proteins, their metabolism and longevity are similar to those of protein-restricted cells. We are currently conducting research on human neurons in this area, and the first result points in the same direction.

 

Not so simple

 

Does this mean that we should change our diet and protein intake? What about other nutrients like sugar? Unfortunately, as expected, things are not that simple. Many other molecules in our body are involved in the perception of nutrients, including carbohydrates, which affect lifespan and age-related diseases.

 

This is why we need to be very cautious. First, everyone’s nutritional needs vary, depending on his/her stage of development, age, gender, or level of activity—only a few important factors are listed here. In addition, although there is growing evidence from the use of human cells and tissues in laboratories, we need to conduct large-scale population studies that record specific diets, including protein, fat, and carbohydrate intake, while analyzing related health or molecular markers. Such research will take decades to produce reliable data and valid conclusions.

 

Nevertheless, with the development of new technologies and scientific methods, we are taking steps to understand the underlying causes of aging and age-related diseases. Coupled with targeted clinical trials and population studies, we may be able to achieve healthy aging and longer life in the near future.

 

Creative Peptides is major supplier of cosmetic peptides, anti-aging peptides for both research and industrial use.

Single-dose Antibody Therapy May Hope to Block HIV Infection and Replication in Newborns

Recently, in a research report published in the international journal Nature Communications, scientists from the Oregon Health and Science University found for the first time that a single dose of antibody-based therapy could be effective in preventing HIV transmission from mother to baby, based on a study of newborn non-human primates.

However, when to give a single dose is the key. The study found that after 30 hours of exposure to SHIV (monkey HIV), newborn cynomolgus monkeys receiving the combination of two antibodies will be prevent from being infected with the virus; if treatment is postponed to 48 Hours, it will cause the other half of newborn cynomolgus monkeys to become infected with SHIV because they received the minimum dose of antibody combination therapy. By contrast, newborn macaques receiving standard HIV therapy (antiretroviral drugs) were protected from SHIV infection when a three-week treatment regimen began 48 hours after exposure. One of the study’s author Dr. Nancy Haigwood said that these promising findings may mean that babies born to HIV-positive mothers can effectively fight HIV infection even with fewer treatments.

In this study, researchers found for the first time in non-human primates that receiving a single dose of a broad-spectrum neutralizing antibody after viral exposure could effectively inhibit SHIV infection. Previous studies have shown that receiving four doses of antibody therapy after exposure can inhibit SHIV infection; in this study, all 10 primate pups were not infected with SHIV within 6 months. Both studies used a combination of two antibodies, PGT121 and VRC07-523. Researchers point out that short-term antiretroviral therapy after viral exposure may effectively inhibit HIV transmission to newborns. Babies born to HIV-positive humans will usually receive a combination of drugs for about 6 weeks, followed by HIV test. If the test result is positive, the babies are required taking HIV drugs for life, but the research in this article shows that starting 3 weeks of antiretroviral therapy 48 hours after exposure may make non-human primates not infected with SHIV.

HIV-positive women usually receive antiretroviral treatment during their pregnancy, which can inhibit the transmission of the virus to developing babies, but sometimes HIV transmission between mothers and babies occurs, and offspring from HIV-positive mothers is usually treated by antiretroviral therapy to suppress viral infections. However, these drug mixtures can also produce many side effects, including special drug formulations for newborns. Researchers also worry about the long-term effects of antiretroviral therapy on the development of children.

Antibodies are non-toxic, and they can be modified and exist in the body for a longer period of time, which can reduce the frequency of patient treatment, so researchers want to study to clarify whether alternative or complementary antiretroviral therapies could be found in the offspring of HIV-positive mothers and adult HIV patients. Next, researchers plan to conduct further research to clarify whether different antibodies or combinations of antibodies and antiretroviral therapy can be more effective. They want to determine whether the antibody therapy they are evaluating can eliminate HIV or inhibit HIV replication.

References:

  1. Shapiro, M.B., Cheever, T., Malherbe, D.C. et al. Single-dose bNAb cocktail or abbreviated ART post-exposure regimens achieve tight SHIV control without adaptive immunity. Nat Commun 11, 70 (2020) doi:10.1038/s41467-019-13972-y

Introduction to Glyco-engineering Antibody

What is glycosylation?

Various modes of action of monoclonal antibodies include neutralization of antigens, complement-dependent cytotoxicity (CDC), and antibody-dependent cytotoxicity (ADCC). Neutralization of antibodies relies on the binding of the Fab region to epitopes on antigens, thereby preventing disease progression due to biomolecular interactions. Monoclonal antibodies acting through CDC and ADCC depend on two stages of action—the first stage: binding of the antibody Fab region to the epitope of the antigen. In the second stage, various types of immune cells can bind to the Fc region of the antibody, thereby triggering the recruitment of “effector” cells in the immune system. These immune cells secrete some cytokines that can ultimately lead to the death of antibody-binding cells. CDC and ADCC involve different death modes of receptors, effector cells and target cells, but both are significantly affected by the type of glycosylation in the Fc functional region of the antibody.

Glycosylation refers to the process or reaction of covalent binding of non-sugar biomolecules to sugar under the action of glycosyltransferases. Glycosylation can be divided into O-linked glycosylation and N-linked glycosylation according to the linkage mode. Due to the differences in the length of the variable region of the antibody, incomplete statistics of the amino acid sequences of the recombinant antibodies published so far revealed that the length of the heavy chain could be between 441 and 454 amino acids, and the glycosylation sites correspondingly varied between Asn-291 and Asn-304.

According to its structure, glycosides can be divided into three types: high mannose type, heterozygous type and complex type. Recombinant monoclonal antibodies can generally contain about 20-30 glycosides, and random pairing of different glycosides of antibody heavy chain can form more than 400 glycoforms. Studies have shown that glycosylation of recombinant monoclonal antibodies can have important effects on antibody-dependent cytotoxicity, complement-dependent cytotoxicity, half-life and immunogenicity of antibody drugs, and can be engineered for glycosylation of recombinant antibody drugs.

Glyco-engineering antibody

 

Figure1. Engineering antibody

Antibody Fc-segment glycans are glycan chains composed of various monosaccharides, such as acetylglucosamine, mannose, galactose, fucose, N-hydroxyacetylneuraminic acid, N-acetylneuraminic acid, etc. Thesemonosaccharides make up polysaccharides through different combinations. Therefore, the antibodies produced by cell fermentation are actually a mixture of antibodies with the same amino acid sequence and different glycan chains. Most of the currently marketed antibody drugs are produced by the expression of CHO cells and NS0 cells. Since these two cells are murine-derived cells, the various glycosylation forms of antibodies expressed by them are exogenous to human. Therefore, these antibody molecules will produce immunogenicity in vivo. Using gene editing technology to alter glycosylation-related genes in antibody-expressing cells can produce glyco-engineering antibodies with higher activity and lower immunogenicity.

  1. Removal of fucose side chains enhances antibody ADCC effect

The majority of Fc-segment glycan chains of recombinant antibodies contain Fuc side chains, and studies have demonstrated that antibodies lacking Fuc have 10 to 100 times the ADCC effect of antibodies with Fuc side chains on glycan chains. Fuc-deleted antibodies can be obtained by overexpressing GMP3 and Golgimannosidase2 glycosylation-related enzymes in host cells, and this modification can greatly improve the activity of antibodies in vivo.

  1. Trigeminal glycan chains enhance antibody ADCC effects

The majority of antibody glycosylation expressed by CHO cells is a bifurcated structure lacking GlcNAc branches. Antibodies glycosylated into trigeminal structures can be obtained by introducing GnTIII enzyme into CHO cells, and antibodies with trigeminal glycosyl chains have higher ADCC activity. Researchers in Switzerland have adapted a host cell for the production of antibodies against neuroblastoma using this technology, and the resulting antibody activity has also been greatly improved.

  1. Overexpression of glycan chain sialic acid can improve the anti-inflammatory activity of antibodies

Fc glycan chains with sialic acid are only a small proportion of recombinant antibodies produced by cell engineering, and the expressed antibodies can be hypersialylated by overexpressing sialyltransferase in host cells. For antibodies used to neutralize in vivo factors, hypersialylation of antibodies can inhibit the immune response of patients after injection of antibodies.

 

What Is Cytomegalovirus and Its Treatments

We rarely hear about this virus in our lives. So, what is cytomegalovirus? How should we prevent cytomegalovirus?

 

What is cytomegalovirus

 

The virus is a herpesvirus DNA virus, also called a cell inclusion virus. Infected cells are swollen and have large intranuclear inclusions.

 

Cytomegalovirus is widely distributed, and other animals can be infected, causing infections in various systems, mainly genitourinary system, central nervous system and liver diseases, from mild asymptomatic infection to severe defects or death.

 

How to prevent cytomegalovirus

 

⑴ Perform conscious physical fitness exercises. Improving the body’s immune function and disease resistance, especially in women of childbearing age, to reduce the serious harm of cytomegalovirus to the fetus.

 

For pregnant women or patients with chronic wasting diseases and low immunity, care should be taken to keep them away from the source of infection.

 

(3) Pay attention to environmental hygiene and diet hygiene.

 

Those who are cytomegalovirus positive in breast milk should not breastfeed.

 

⑸Immune control is currently under study. After causing cell infection, inactivated vaccine has no obvious preventive effect. If an infection is found early in pregnancy, the pregnancy should be terminated.

 

Live attenuated vaccines allow the recipient to produce antibodies. It also produces cellular immunity to CMV and reduces the occurrence of symptomatic cmv infection. CMV high-valent immunoglobulin has a protective effect on symptomatic CMV infection in serum CMV-negative bone marrow transplant recipients, but it cannot prevent reinfection.

 

Wash hands carefully after contact with urine or saliva of children to prevent acquired CMV infection.

 

To prevent CMV infection caused by fresh blood transfusion, the following methods can be used: ① Use frozen or washed blood; ② Store blood for more than 48 hours before transfusion; ③ Use radiation-exposed blood; ④ Use a blood filter to remove blood from the blood. Giant cells.

 

CMV treatment

 

The infection of this virus is mostly in a latent infection state. In time, he replicates and activates in the human body, and most of them are asymptomatic infections. Therefore, its treatment is limited to symptomatic infections.

 

General treatment

 

CMV hepatitis can be used in accordance with the general principles of treating viral hepatitis to promote liver cell recovery, reduce liver cell damage, reduce aminotransferases, and resolve jaundice. Infusion of albumin if necessary. Those who are severely smoked with anemia will be transfused as appropriate. CMV pneumonia is symptomatic and supportive of viral pneumonia.

 

Antiviral therapy

 

Ganciclovir is currently the drug of choice in anti-HCMV treatments. Commonly used intravenously. 7.5-10mg / (kg · d), divided into 2 to 3 intravenous drips, 14 days later changed to 5 mg / (kg · d) once intravenously, maintained for 1-2 months. This drug inhibits the DNA polymerase activity of HCMV, affects the DNA synthesis of the virus, and thus inhibits the virus.

 

Common adverse reactions of this drug are leukocytes (mainly neutrophils) and thrombocytopenia, which can be recovered after stopping the drug. Sodium phosphonate is highly selective for HCMV, acts on viral DNA polymerase, and inhibits HCMV replication. The main side effect is kidney damage, and the inhibitory effect on bone marrow is less than GCV. The dose is 150 mg / (kg 3 weeks.

 

Those who are ineffective for GCV treatment can switch to this medicine. Severe cases can be combined with the two drugs mentioned above, ganciclovir 35 mg / (kg · d) and sodium phosphonate 60 mg / (kg · d). It can also be used alternately with ganciclovir 5mg / (kg · d) and sodium phosphonate 90mg / (kg · d).

 

Other antiviral drugs such as adenosine arabinoside, acyclovir, zidovudine, and interferon all have the effect of inhibiting HCMV, but cannot clear HCMV infection.

A Huge Leap of the mAb Drug Against Tumor—Humanized Monkey Antibody

It’s a common sense among the professionals working in the field of anti-tumor medicine that the antibody drugs outperform traditional ones in efficacy on a large scale, of which the humanized mAbs are a pillar rock in midstream.

 

Overview of antibody humanization

Original mAb drugs come from immunization in mice, like Muromonab-cd3, the first new monoclonal antibody drug approved by FDA in history, which have high immunogenicity and are liable to induce human body produce corresponding antibodies, and thus neutralized the drugs themselves. As a result, since the 1980s, scientists have been dedicated to cutting down immunogenicity in the mAbs by increasing the proportion of sequences from human body in the antibody sequence, which was the first attempt of humanization. Yet the humanized antibody whose host is rodent still can not achieve satisfactory efficacy as there are still alloplasmic sequences. As technology advances, methods in this stream have evolved too. Creative Biolabs, as a conscientious CRO in the field of antibody humanization, has worked out a way to humanize antibodies from monkey (non human primate, NHP), leading the whole industry.

 

The notion of adopting monkeys’ antibody originates from their close relationship with human beings, and the unsatisfactory test results of rodent animals. In order to accelerate the research steps in the whole field, a humanization platform has been designed that accepts not simply rabbit, dog, chicken, but also monkey antibodies for humanization, on which two crucial NHP’s 3D antibody prototypes are built so as to detect mutations.

 

Major techniques adopted during the NHP antibody humanization process:

 

CDR Grafting & SDR Grafting—complementarity-determining region grafting combined with specificity-determining residue grafting to ensure lessening the immunogenicity of the resulting antibody as much as possible, even in the residues.

 

Chain Shuffling Strategybased on construction and screening of two chimeric phage display libraries to orderly shift the light chain and heavy chain of antibody for further screening, which enables full humanization.

 

Humanized IgG Library Screening—a mammalian cell surface display approach allowing selecting humanized antibodies in a full-size IgG format, and support protein synthesis.

 

NHPs are involved in the study of more than 70 kinds of human infectious diseases, including pathogens like bacteria, viruses, fungi, parasites and prions; infectious diseases like global infectious diseases, children’s infectious diseases, tropical infectious diseases, sexually transmitted diseases, new infectious diseases, zoonoses, potential bioterrorism, etc. Creative Biolabs’s NHP humanization solutions can solve most of the problems encountered by researchers and medicine manufactures that large-scale experiments on NHPs are costly and confined to high experimental standards, that systematic fundamental immunological information of NHPs’ gene, protein metabolites and signaling pathways are lacked. Furthermore, the application of NHP humanized antibodies in immunotherapy is hopeful to become more common to fix with the high risk of immune reaction stimulated by humanized rodent antibodies. More details can be reached at https://www.creative-biolabs.com/.

Fitness Boot Camp Marketing – How to Tips

Every marketer’s dream is to come up with new and exciting gimmick that they can market, for that particular industry. Hence, when good fitness boot camp marketing becomes available and can be marketable, it would surely be noticed by almost every marketer and individual in business or those whose interest lies not just in gaining profit but to just plainly lose weight without bothering to do it regularly.
So what does constitute an ideal fitness boot camp marketing to be considered marketable? This is what you need to know as a marketer to be able to use this with other fitness boot camp that is desperately seeking special help to boast income fast. So that spells more money for you, the trainer and good health for the fitness trainee.
Just keep in mind that a marketable fitness camp marketing is one that could make money for you and the boot camp trainer in less than a few hours over a period of time, and still have more time to spend for the other areas of their life. This is because their training time is more or less around 45 minutes only for group of 7- 20 individuals.
So here are the criteria for excellent and marketable fitness camp marketing, one that could bring loads of money.
Create an ideal and exciting workouts – The keyword on this is “fun” because the fitness camp marketing most project an image that the camp is exciting and worth joining. In any business it is all about projection, as what you tell your future clients is very important in their decision making.
Therefore, if your projection is somewhat a fun camp that does not entail only pure workouts, but also in having a great time aside from losing their excess pounds for good, is what will surely make your camp profitable.
Positive Results – An already existing boot camp has major advantage over newly opened camp and one of them is “proven results.” Existing camp can already tell other people what they have done or achieved, additionally they will have people give out true testimony on how the camp help were able to help them get back into shape. In case you do not know, word of mouth is the most fastest and effective way to gain the awareness of everyone.
Free Trials – This is a standard but totally cool fitness boot camp marketing tactics. Let us just admit it, everyone loves freebies and if anybody offer one then they can expect potential paying clients lining up to sign-up for the program.
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